Human cytomegalovirus (HCMV) is an important human pathogen that causes severe morbidity and mortality in the immunocompromised and immunosuppressed individual. The virus has the ability to cross the placenta and infect the fetus in utero which can lead to severe congenital abnormalities and birth defects. Human cytomegalovirus (HCMV) gene expression is a complex, coordinated process consisting of three phases, immediate early (IE), early and late. IE proteins regulate viral and cellular gene expression and are important for activation of early promoters. Specifically, IE1 and 2 act synergistically to activate early promoters. Studies have demonstrated that other IE genes (UL36-38, IRS1 and TRS1) as well as at least two early gene products (UL69 and UL112- 113) function to enhance IE1 and 2 mediated activation of early promoters. The early phase of the virus life cycle is necessary to prepare the cell for virus DNA replication. It is generally thought that if the virus proceeds through the early phase and properly initiates virus DNA replication, then the infection will result in the generation of infectious progeny. Therefore, the early phase of virus DNA replication is thought to be a commitment point for the successful completion of infectious progeny production. In this proposal, we will conduct studies to address how vital transacting proteins, and in particular how IE1 and 2, UL112-113 and IRS1/TRS1 proteins regulate viral early promoters. The promoters chosen, pol (UL54), UL44 and ICP8 (UL57) are members of the 11 loci shown to be important for viral DNA replication. We will 1) identify those transacting regulatory proteins which influence early promoter activity, 2) identify domains within those proteins which carry out this effect, 3) identify the sequences within the early promoters responsive to viral proteins which augment IE1 and 2 mediated activation of early promoters and characterize the proteins that interact with those sequences, 4) characterize the interaction of IE proteins with the pol promoter and the basal transcription factor complex and 5) test the biological relevance of the identified promoter elements in the context of the viral genome and to use these recombinant viruses to begin to address cell type specific differences in the regulation of HCMV early promoters.